Since establishing the Molecular and Cell Biology group in August 2008, lab personnel have been hired and significant progress made in acquiring preliminary data for computational simulations of TCR-mediated Erk activation. We have established protocols in the lab for isolation and purification of CD4+ T cells from transgenic mice (5CC7/Rag2) that express T cells against a defined ligand (PCC). In collaboration with Rajat Varmas laboratory, we have acquired a method for generating bead-based lipid bilayers containing the appropriate peptide-loaded MHC and co-stimulatory molecules to provide a reproducible cell-free stimulus to the 5CC7/Rag2 CD4+ T cells. This is important as activation with intact APCs would complicate measurements of Erk cascade protein concentrations and phosphorylation as the cell lysates would contain a mixture of these proteins from both T cell and APC sources. We have generated protein standards for quantificatation of Raf1, Mek1+2, Erk1+2 and Ksr by expressing the murine cDNAs as tagged fusions in bacterial and/or mammalian cells. The optimal expression and purification method has been identified in each case to give acceptable quantities of pure protein for loading reference samples on quantitative western blots. Multiple commercial antibody sources for these targets have been assessed to identify suitable antisera for both western and flow cytometry measurements. We have also assessed the suitability of the xMAP (multi-analyte profiling) technology developed by Luminex for quantitative assessment of protein concentration and phosphorylation. So far, this has provided high quality data for several components of the cascade. We intend to use all of the above approaches to increase confidence in our estimations of both protein number and degree of protein phosphorylation in response to stimulus. Thus far, the data we have obtained for the Mek and Erk proteins expressed in 5CC7/Rag2 CD4+ T cells by both xMAP and quantitative western blotting have shown good agreement. We are currently working on validation of suitable antisera for flow cytometry detection of the Erk cascade proteins. This is important as it provides single cell data and an indication of the population distribution of protein concentrations and phosphorylation responses. We are also developing shRNAs against each target to permit calibration of the quantitative expression data by comparing cells with and without RNAi-based depletion of each Erk cascade protein. Another key requirement in this project is to acquire an estimation of the morphology and geometric characteristics of the T cell to allow accurate calculations of the concentrations of the signaling proteins in the cell. This has been achieved through staining the 5CC7/Rag2 CD4+ T cells with selective dyes and expression of a panel of subcellular markers that localize to specific regions of the cell. In collaboration with the NIAID RTB imaging core laboratory, we have used confocal microscopy and 3D cell reconstruction software to generate reproducible data on cell shape characteristics and volume.